ES2728305T3 - Use as an O-acetylated GD2 ganglioside target as a new therapeutic and diagnostic strategy for cancer stem cell cancer - Google Patents

Abstract

A pharmaceutical composition for use in the treatment of breast cancer comprising cancer stem cells that have at least one phenotype chosen from the group comprising or consisting of CD44 + / CD24- / low, CD44 + / CD24- / low / ESA + or CD44 + / CD24- / bajo / lin- / ALDH1 +, said pharmaceutical composition comprising an antibody that recognizes the O-acetylated GD2 ganglioside or one of its functional fragments, and wherein said antibody comprises: a) a light chain comprising at least one frame of light chain variable region of an immunoglobulin and three complementarity determining regions (CDR) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO: 3 for CDR -L3, and / or b) a heavy chain comprising at least one heavy chain variable region framework of an immunoglobulin and three complementarity determining regions (CDR) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, SEQ ID N Or: 6 for CDR-H3.

Description

DESCRIPTION

Use as a target of the O-acetylated GD2 ganglioside as a new therapeutic and diagnostic strategy for cancer stem cell cancer

Field of the Invention

The present invention relates to antibodies, pharmaceutical compositions and their use in methods for treating and / or diagnosing cancer of cancer stem cells (hereinafter referred to as CSC, by the English expression Cancer Stem Cells).

Background of the invention

Cancer stem cells

The advances in medical research in these last decades allowed to significantly improve the existing strategies in cancer therapy. Parallel to the continuous study and adaptation of conventional therapeutic strategies, including chemotherapy, radiotherapy, hormonal therapy and surgery, new approaches to the treatment of cancer appeared, called "targeted anti-cancer therapies" and demonstrated their effectiveness and interest in the world doctor. These targeted therapies include monoclonal antibodies directed against tumor antigens, such as trastuzumab (anti-Her2Neu), rituximab (anti-CD20) or bevacizumab (anti-VEGF), but also tyrosine kinase inhibitors (for example, Imatinib, Erlotinib, Gefitinib ), CDK inhibitors, etc. These new solutions for cancer treatment greatly improved the lives of patients with a higher survival rate and lower side effects compared to more conventional strategies. However, these targeted therapy strategies also quickly show limits, particularly due to the resistance to such treatments, but also to the specificity of these targeted therapies, which are not effective in all types of cancer and patients. These resistance phenomena lead to treatment failure, metastasis, relapse and relapse.

Studies have shown that the root of these resistances could be the presence of cancer stem cells (CSC), a small subgroup of cancer cells that have the ability to self-renew and differentiate, and play a key role in stubborn cancer recurrence. and metastasis. In fact, these cancer stem cells show resistance relative to cancer treatments, including radiation and chemotherapy. In addition, only a small amount of these cancer stem cells is enough to start a new tumor and, therefore, metastasis. Therefore, a direct use of these cancer stem cells as a target can improve the effectiveness of current cancer therapy strategies.

Cancer stem cells and associated mechanisms have been studied extensively. Methods to identify these cancer stem cells have been investigated and few CSC markers were found. Unfortunately, there is not yet an antigen identified as a marker of CSCs that is useful in many types of tumors. Examples of CSC markers currently used include ALDH, CD133, CD44, CD24, CD166. Many different phenotypes have been identified as CSC phenotypes in different types of cancers. In particular, CD133, which is now widely used as a marker of cancer stem cells, appears to be a good marker of CSC in the glioma.

Today, the CD133 + cell phenotype is related to cancer stem cells in the glioma; the CD44 + CD24 '/ low phenotype appears to be related to cancer stem cells in breast cancer; The CD34 + phenotype appears to be related to cancer stem cells in leukemia, more particularly, CD34 + / CD38 is related to cancer stem cells in acute myeloid leukemia and the CD34 + / CD19 + phenotype appears to be related to cancer stem cells in acute lymphoid leukemia.

Unfortunately, most known CSC markers are also expressed in some normal tissues. Therefore, it would be of great interest to identify specific cancer markers that could be used as diagnostic and therapeutic CSC markers: they could be used as diagnostic targets, but also to treat cancers comprising cancer stem cells, referred to herein as cancer of cancer stem cells (CSC cancer), without significant side effects due to toxicity to healthy cells.

Summary of the invention

The inventors have already shown that cancers of neuroectodermal origin specifically express the G-2 O-acetylated ganglioside and that a therapeutic antibody directed to the G-2 O-acetylated ganglioside can be administered and show beneficial effects without neurotoxicity, especially due to the absence of expression of this antigen Carcinogenic in healthy cells, especially in peripheral nerves.

To this end, the inventors created an antibody that recognized the GD2 O-acetylated ganglioside and did not recognize the GD2 ganglioside, for use in the treatment of cancers expressing O-acetylated GD2, in particular neuroblastomas, melanomas, glioblastomas or lung cancers of small cells (document FR 2906 808 Now and surprisingly, the inventors show that GD2 O-acetylated ganglioside expression is enriched in cancer stem cells and could be a target for the treatment of CSC cancer.

Thus, the inventors propose that the O-acetylated GD2 ganglioside be a target for the treatment of CSC cancer, using the 8B6 antibody (and its derivatives) which is specific for the O-acetylated GD2 ganglioside. In their studies, they demonstrate that this antibody shows potent intrinsic cytotoxic activity against tumor cells, including direct cytotoxicity to cancer stem cells through apoptosis or other cell death pathways, in addition to immune effects, including CDC and ADCC .

Therefore, the present invention relates to an antibody that recognizes the O-acetylated GD2 ganglioside, or one of its functional fragments, for the treatment of cancer stem cell cancer (CSC), said antibody comprising:

a) a light chain comprising at least one light chain variable region framework of an immunoglobulin and three complementarity determining regions defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2 , SEQ ID NO: 3 for CDR-L3, and / or

b) a heavy chain comprising at least one heavy chain variable region framework of an immunoglobulin and three complementarity determining regions defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2 , SEQ ID NO: 6 for CDR-H3.

The invention also relates to a pharmaceutical composition for use in a method of treating CSC cancer, said pharmaceutical composition comprising an antibody of the invention.

The present invention further relates to a method for diagnosing a CSC cancer in a subject, wherein said method comprises determining the expression of the O-acetylated GD2 ganglioside, and wherein an O-acetylated GD2 ganglioside expression is indicative of a CSC cancer

The invention also relates to the use of the G-2 O-acetylated ganglioside as a biomarker of CSC cancer. The invention finally relates to a method for predicting the response of a subject affected with CSC cancer to a treatment with an antibody or a composition of the invention, wherein said method comprises detecting the presence of cells expressing the ganglioside GD2 O- acetylated in a biological sample of said subject. Description of the drawings

Figure 1: Representative images of a glioblastoma tumor section stained with the control IgG3 antibody as a negative control (A); Neuroblastoma tumor stained with mAb 8B6 (B). Glioblastoma tumor samples were scored 1+ (C), 2+ (D), 3+ (E) and 4+ (E) according to staining with mAb 8B6 of specific intensity.

Figure 2: OAcGD2 identifies the phenotype of CD133 + stem cells in human glioblastoma cancer cells. UL, upper left quadrant, human glioblastoma cells OAcGD2 + U87MG; UR, right upper quadrant, CD133 + OAcGD2 + U87MG human glioblastoma cells; LL, left lower quadrant, human glioblastoma cells CD133 'OAcGD2 lio U87MG; LR, right lower quadrant, CD133 + U87MG human glioblastoma cells.

Figure 3: Microscopic contrast test of U87MG human glioblastoma cells phases exposed to 50 pg / mL of control antibody or 8B6 mAb. After an incubation period of 24 hours, apoptotic cells evaluated by morphological changes were observed under a phase contrast microscope with the same magnifications 200. The arrow indicates apoptotic cell.

Figure 4: The anti-OAcGD2 mAb inhibits the growth of human glioblastoma tumors in mice.

Figure 5: Cytotoxicity of anti-GD2 and anti-OAcGD2 antibodies in breast cancer cells. Direct cytotoxicity is evaluated by the MTT assay with increasing concentrations of anti-GD2 (10B8) and murine anti-OAcGD2 (8B6) antibodies.

Figure 6: Expression of OAcGD2 in CSC in small cell lung cancer. Expression profiles in flow cytometry for the H196 cell lines of CD133 (CSC) simultaneously with OAcGD2 (8B6) or GD2 (10B8).

Detailed description of the invention

Therapeutic strategies of the invention

The first object of the invention relates to an antibody that recognizes the G-2 O-acetylated ganglioside, or one of its functional fragments, for the treatment of cancer of cancer stem cells (CSC), said antibody comprising:

a) a light chain comprising at least one light chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO 3 for CDR-L3, and / or

b) a heavy chain comprising at least one heavy chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, SEQ ID NO: 6 for CDR-H3.

Such CDRs are defined according to the IMGT nomenclature that is well known in the art (The International Immunogenetics Information System®, LEFRANC et al., Nucleic Acids Research, vol. 27, p: 209-212, 1999).

The term "antibody" has its general meaning in the art and refers to an immunoglobulin molecule corresponding to a tetramer comprising four polypeptide chains, two identical (H) heavy chains (approximately 50-70 kDa when full length) and two identical light chains (L) (approximately 25 kDa when full length) interconnected by disulfide bonds. Light chains are classified as kappa and lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and define the antibody isotype as IgG, IgM, IgA, IgD and IgE, respectively. Each heavy chain is composed of a variable region of the heavy chain at the N-terminus (abbreviated here HCVR) and a constant region of the heavy chain. The constant region of the heavy chain comprises three domains (CH1, CH2 and CH3) and a hinge region for IgG, IgD and IgA; and 4 domains (CH1, CH2, CH3 and CH4) for IgM and IgE. Each light chain is composed of a variable region of the light chain at the N end (abbreviated in this document LCVR) and a constant region of the light chain. The constant region of the light chain is composed of a domain, Cl. The HCVR and LCVR regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each HCVR and LCVR is composed of three CDR and four FR, arranged from the amino end to the carboxy end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with well-known conventions. The functional ability of the antibody to bind to a particular antigen depends on the variable regions of each pair of light / heavy chains, and is largely determined by the CDRs.

In a particular embodiment, the antibody of the invention comprises:

a) a light chain comprising a light chain framework of an immunoglobulin light chain and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2 , SEQ ID NO: 3 for CDR-L3, I

b) a heavy chain comprising a heavy chain framework of an immunoglobulin heavy chain and three complementarity determining regions ( ^c D ^r ) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, SEQ ID NO: 6 for CDR-H3.

In a more particular embodiment, the antibody of the invention comprises a light chain variable region (LCVR) comprising the amino acid sequence SEQ ID NO: 7 and a heavy chain variable region (HCVR) comprising the amino acid sequence SEQ ID NO: 8. Said antibody is antibody 8B6.

The term "antibody", as used herein, refers to a monoclonal antibody per se. A monoclonal antibody can be a human antibody, a chimeric antibody and / or a humanized antibody.

The term "functional fragment," as used herein, refers to an antibody fragment capable of recognizing the O-acetylated GD2 ganglioside. Such fragments can be identified or produced simply by the person skilled in the art and comprise, as an example, a Fab fragment (which can be produced by papain digestion), a Fab 'fragment (which can be produced by pepsin digestion and partial reduction ), an F (ab ^{') 2} fragment (which can be produced by digestion with pepsin), a Facb fragment (which can be produced by digestion with plasmin), but also a scFv fragment (single chain Fv, produced by techniques of molecular biology), diabody and monobody.

The term "diabody" refers to small antibody fragments with two antigen binding sites, said fragments comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH) - VL). Preferably, using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to mate with the complementary domains of another chain and create two antigen binding sites.

The term "monobodies", as used herein, refers to antigen-binding molecules with a heavy chain variable domain and no light chain variable domain. A monobody can bind to an antigen in the absence of a light chain and typically has three CDR regions designated CDRH1, CDRH2 and CDRH3. Monobodies include "camelid monobodies," such as VHH fragments obtained from an animal source of the camelid family, including animals with two-toed legs and leather plants. Animals in the family of camelids include camels, llamas and alpacas. It has been reported that camels ( Camelus dromedaries and Camelus bactrianus) often lack variable light chain domains when analyzing their serum material, suggesting that sufficient specificity and affinity of antibodies can be derived from domains VH (three loops of c Dr) alone. Monobodies also include modified VH from various animal sources, particularly mammals (eg, mouse, rat, rabbit, horse, donkey, bovine or human), which can bind to an antigen in the absence of VL. Preferably, the VH is modified at positions on the VL interface to provide VH binding to the antigen in the absence of the VL. One skilled in the art is able to optimize a human VH by replacing some important residues (by "camelization") to mimic heavy chains of camelid antibodies naturally devoid of light chain partners. This allows to obtain functional antibody fragments with stability properties and expression levels similar to those of camelid VHH, while maintaining the recognition properties of the antibody fragment, which include high affinity and high specificity and decreased immunogenicity.

Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as is known in the art and / or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a heavy chain portion F (ab ^{') 2} can be designed to include DNA sequences encoding the CH1 domain and / or the hinge region of the heavy chain. The various portions of antibodies can be chemically bound by conventional techniques or can be prepared as a contiguous protein using genetic engineering techniques.

In a particular embodiment, the invention relates to a functional fragment of an antibody of the invention in which said fragment is chosen from the group comprising or consisting of Fab, Fab ', F (ab') ² , Facb, Fd, scFv, diabodies and monobodies that include VHH fragments and human VH fragments.

According to the invention, said functional fragment is capable of recognizing the G-2 O-acetylated ganglioside. Preferably a functional fragment of the antibody of the invention retains an equivalent activity, particularly an equivalent cytotoxic activity, to that of an antibody of the invention.

The expression "recognize the O-acetylated GD2 ganglioside" means that the antibody of the invention is capable of binding to the O-acetylated GD2 ganglioside with an affinity of less than 10'7 M, preferably less than 5 x 10'8 M and more preferably less than 10'8 M.

Preferably, but not necessarily, the antibodies useful in the invention are produced recombinantly, since the manipulation of typically murine or other non-human antibodies with the appropriate specificity is required to convert them into humanized form. Antibodies may or may not be glycosylated, although glycosylated antibodies are preferred. The antibodies are suitably crosslinked by disulfide bonds, as is well known.

As is well understood in the art, monoclonal antibodies can be easily generated with an appropriate specificity by standard mammalian immunization techniques that produce it. These nucleotide sequences can be manipulated to provide them in humanized form.

By "chimeric antibody" is meant an antibody that is composed of variable regions of a murine immunoglobulin and constant regions of a human immunoglobulin. This alteration simply consists in replacing the murine constant region with the constant region of a human antibody, which results in a human / murine chimera that can have an immunogenicity sufficiently low to be acceptable for pharmaceutical use.

Until now, various methods for producing such chimeric antibodies have been described, which are part of the general knowledge of the person skilled in the art (see, for example, US Patent No. 5,225,539). By "humanized antibody" is meant an antibody that is partially or totally composed of amino acid sequences derived from a germ line of a human antibody, altering the sequence of an antibody having non-human complementarity determining regions (CDRs). This humanization of the variable region of the antibody and, finally, the CDR is performed by methods that are now well known in the art.

As an example, British Patent Application GB 2188638A and United States Patent No. 5,585,089 describe processes in which recombinant antibodies are produced in which the only portion of the antibody that is substituted is the complementarity determining region, or "CDR". The CDR grafting technique has been used to generate antibodies consisting of murine CDRs, and the framework of the human variable region and constant regions (see, for example, RIECHMANN et al., Nature, vol.332, p: 323-327, 1988). These antibodies retain the human constant regions that are necessary for Fc-dependent effector function, but are much less likely to elicit an immune response against the antibody.

As an example, the frame regions of the variable regions are replaced by the corresponding human frame regions leaving the non-human CDR substantially intact, or even replacing the CDR with sequences derived from a human genome (see, for example, the application publication US Patent 2006/0258852 A1). Fully human antibodies are produced in genetically modified mice whose immune systems have been altered to correspond to human immune systems. As mentioned above, it is sufficient for use in the methods of the invention to employ an immunologically specific fragment of the antibody, including fragments that represent single chain forms.

A humanized antibody again refers to an antibody comprising a human framework, at least one CDR of a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, that is, at least about 85 or 90%, preferably at least 95% identical. Therefore, all parts of a humanized antibody, except possibly the CDRs, are substantially identical to the corresponding parts of one or more natural human immunoglobulin sequences. For example, a humanized immunoglobulin would typically not encompass a mouse variable region / human constant region chimeric antibody.

Humanized antibodies have at least three potential advantages over nonhuman and chimeric antibodies for use in human therapy:

1) Because the effector portion is human, it can better interact with the other parts of the human immune system (for example, destroy target cells more efficiently by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) )).

2) The human immune system should not recognize as foreign the framework or region C of the humanized antibody, and therefore the response of the antibody against said injected antibody must be less than against a totally foreign non-human antibody or a chimeric antibody partially strange

3) It has been reported that injected non-human antibodies have a half-life in human circulation much shorter than the half-life of human antibodies. Injected humanized antibodies will have a half-life essentially identical to naturally occurring human antibodies, allowing smaller and less frequent doses to be administered.

As an example, the design of humanized immunoglobulins can be carried out as follows: when an amino acid is within the following category, the framework amino acid of a human immunoglobulin to be used (acceptor immunoglobulin) is replaced by an amino acid of a non-human immunoglobulin framework that provides the CDRs (donor immunoglobulin): (a) the amino acid in the human framework region of the acceptor immunoglobulin is unusual for human immunoglobulin in that position, while the corresponding amino acid of the donor immunoglobulin it is typical of human immunoglobulin in that position; (b) the position of the amino acid is immediately adjacent to one of the CDRs; or (c) any side chain atom of a framework amino acid is within approximately 5-6 A (center to center) of any atom of an amino acid of a CDR in a three-dimensional immunoglobulin model (QUEEN et al., Proc. Natl. Acad. Sci. USA, vol. 88, p: 2869, 1991). When each of the amino acids in the human frame region of the acceptor immunoglobulin and a corresponding amino acid in the donor immunoglobulin is unusual for human immunoglobulin in that position, said amino acid is replaced by a typical amino acid of the human immunoglobulin in that position.

In a particular embodiment, the antibody of the invention is a chimeric antibody.

In a preferred embodiment, said antibody is a chimeric antibody and the framework sequences of the light and heavy chains are from the light and heavy chains of mouse immunoglobulin, respectively.

Preferably, said chimeric antibody further comprises the constant regions of human light and heavy chains.

According to the invention, a chimeric antibody of the invention is capable of recognizing the G-2 O-acetylated ganglioside.

Preferably, a chimeric antibody of the invention retains an equivalent activity, particularly an equivalent cytotoxic activity, to that of an antibody of the invention.

Advantageously, said antibody comprises a light chain variable region (LCVR) comprising the amino acid sequence SEQ ID NO: 9.

Again advantageously, said antibody comprises a heavy chain variable region (HCVR) comprising the amino acid sequence SEQ ID NO: 10.

In a more preferred embodiment, said antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO: 9 and a heavy chain variable region (HCVR) comprising the amino acid sequence SEQ ID NO: 10.

In another particular embodiment, the antibody of the invention is a humanized antibody.

In another preferred embodiment, said antibody is a humanized antibody and the framework sequences of the light and heavy chains are derived from the light and heavy chains of humanized immunoglobulin respectively. Preferably, a humanized antibody of the invention further comprises the constant regions of the human light and heavy chains.

According to the invention, a humanized antibody of the invention is capable of recognizing the G-2 O-acetylated ganglioside.

Preferably, a humanized antibody of the invention retains an equivalent activity, particularly an equivalent cytotoxic activity, to that of an antibody of the invention.

Other sequences are possible for light and heavy chains for humanized antibodies of the present invention. The immunoglobulins can have two pairs of light chain / heavy chain complexes, at least one chain comprising one or more mouse complementarity determining regions functionally linked to the segments of the human frame region.

The antibodies of the invention encompass immunoconjugates.

As used herein, the term "immunoconjugate" refers to a conjugated molecule comprising at least one antibody or one of its functional fragments, attached to a second molecule, preferably a cytotoxic agent or a radioisotope. Preferably, said antibody or one of its functional fragments is linked to said second molecule by covalent bond.

In one embodiment, the antibody of the invention is an immunoconjugate.

In a particular embodiment, the antibody of the invention is an immunoconjugate, wherein said immunoconjugate comprises an antibody of the invention or one of its functional fragments and a cytotoxic agent.

In another particular embodiment, the antibody of the invention is an immunoconjugate in which said immunoconjugate comprises an antibody of the invention or one of its functional fragments and a radioisotope. The term "cancer stem cell cancer" or "CSC cancer" refers to a cancer that comprises particular cells known as cancer stem cells.

In one embodiment, a CSC cancer comprises at least 0.1% of cancer stem cells; preferably, a CSC cancer comprises 1% to 95% of cancer stem cells, more preferably a CSC cancer comprises 1% to 50% of cancer stem cells

The term "cancer stem cells" has its general meaning in the art and refers to a subpopulation of cancer cells (found within solid tumors and hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability of giving rise to all types of cells found in a particular cancer sample. They have the capacity for self-renewal, differentiation in multiple lineages of cancer cells and extensive proliferation. They can start a new tumor with only a small amount of cancer stem cells and tend to be resistant to conventional therapy, including chemotherapy and radiation therapy.

Therefore, using the CSC as a target, the antiOAcGD2 antibody allows to prevent and / or treat the resulting metastasis of these CSCs.

These cancer stem cells can be isolated from tumors by surface markers, such as CD133, CD44, CD34, CD24, ALDH1; These markers make it possible to distinguish cancer stem cells from most cancer cells. Several methods are currently known in the art to specifically isolate cancer stem cells in vitro, including specific markers of widely used methods or Hoechst stain-based isolation, but also chemo-resistance-based isolation, heterogeneity of classification of invasiveness and, in metastatic breast cancer cells, classification by reoxygenation after exposure to repetitive cycles of hypoxia.

Cancer stem cells have been identified in very different types of cancers, including, but not limited to, leukemia that includes acute myeloid leukemia and acute lymphoid leukemia, breast cancer, glioma that includes glioblastoma, colorectal cancer, pancreas cancer, prostate cancer , lung cancer, liver cancer, bladder cancer or gastric cancer.

Therefore, in one embodiment of the invention, the CSC cancer of the invention is chosen from the group comprising or consisting of leukemia that includes acute myeloid leukemia and acute lymphoid leukemia, breast cancer, glioma that includes glioblastoma, colorectal cancer , pancreatic cancer, prostate cancer, lung cancer, liver cancer, bladder cancer or gastric cancer.

In a preferred embodiment, said CSC cancer is glioma, breast cancer, acute lymphoid leukemia or acute myeloid leukemia.

Different markers that identify cancer stem cells have been observed among most cancer cells, such markers varying and depending on the type of cancer.

Examples of markers that can be used to identify CSC cancer include, but are not limited to, CD34, CD38, CD19, interleukin-3-receptor a (CD123), CD33, CD44, CD44v6, CD47, CD24, EpCAM (ESA) , Lin, CD133, A2B5, SSEA-1, CD166, CD26, CD200, a2pi, Sca, CD45, Pecam, ALDH, ALDH1, Oct4, ABCG2, CXCR4, AFP, EMA, IGF-IR.

However, it should be borne in mind that known cancer stem cell markers can be used to identify CSC cancer, but they are not suitable as a target antigen to treat said cancer, since they are expressed primarily in healthy tissues.

Some examples of cancer stem cell phenotypes include the phenotypes described in Table 1:

Table 1: Biomarkers and / or phenotypes of different cancer stem cells

Therefore, in one embodiment, said CSC cancer comprises cells that have at least one phenotype chosen from the group comprising or consisting of: CD34 + / CD38-, CD34 + / CDl9-, CD34 + / CD19 +, CD34 + / CD38 + / CD19 +, CD34 + / CD38- / CD19 +, CD133 + / CD38-, CD133 + / CD19-, inerleukin-3-receiver a +, CD33 +, CD44 + / CD24- / bass, CD44 + / CD24- / ESA +, CD44 + / CD24 under / -linVALDH +, CD133 +, A2B5 + , SSEA-1 +, CD133 + / ESAalto / CD44 +, CD166 +, CD26 +, CD44 + / CD133 + / a2p1 +, CD44 +, Sca + / CD45- / Pecam- / CD34 +, ALDH1 + / Oct4 + / CD133 + / ABCG2 + / CXCR4 +, CD133 + / CD44 + / CD44 + / CD44 + / CD44 + / CD44 + AFP +, EMA- / CD44v6 +, CD133 + / CD44 + / ALDH1 +.

In one embodiment of the invention, the CSC cancer of the invention comprises CD133 + cells.

In another embodiment, the CSC cancer of the invention comprises CD44 + cells. In a more particular embodiment, the CSC cancer of the invention comprises CD44 + / CD24- / low cells.

In yet another embodiment, the CSC cancer of the invention comprises CD34 +

In a more particular embodiment, the CSC cancer of the invention comprises CD34 + / CD38- cells.

In another more particular embodiment, the CSC cancer of the invention comprises CD34 + / CD19 + cells, preferably CD34 + / CD38 + / CD19 + or CD34 + / CD38- / CD19 + cells

In a preferred embodiment of the invention, a CSC cancer of the invention is characterized by a subpopulation of cancer stem cells in which at least 10% of the cancer stem cells have the O-acetylated GD2 ganglioside on their surface, preferably at minus 30% of cancerous stem cells, and more preferably at least 50% of the cancerous stem cells have the O-acetylated GD2 ganglioside on their surface.

In a particular embodiment of the invention, said CSC cancer is breast cancer.

In a more particular embodiment, said CSC cancer is breast cancer in which cancer stem cells are characterized by a CD44 + / CD24 '/ low phenotype.

In another particular embodiment of the invention, said CSC cancer is glioma.

In a more particular embodiment, said CSC cancer is a glioma in which cancer stem cells are characterized by a CD133 + phenotype.

In another particular embodiment of the invention, said CSC cancer is leukemia, more particularly acute myeloid leukemia or acute lymphoid leukemia.

In a more particular embodiment, said CSC cancer is leukemia where the cancer stem cells are characterized by a CD34 + phenotype, more particularly said CSC cancer is acute myeloid leukemia where the cancer stem cells are characterized by a CD34 + / CD38- phenotype. or acute lymphoid leukemia where the stem cells are characterized by a CD34 + / CD19 + phenotype, preferably a CD34 + / CD19 + / CD38- or CD34 + / CD19 + / CD38 + phenotype.

In the context of the invention, the term "treating CSC cancer" means reversing, alleviating, inhibiting the progress or spread of CSC cancer and / or metastasis. The term also covers the prevention of CSC cancer, metastasis or recurrence of CSC cancer. Preferably, such treatment also leads to regression of tumor growth, that is, the decrease in the size of a measurable tumor. Typically, such treatment leads to a prolongation of the patient's survival without progression of CSC cancer, recurrence or a prolongation of their overall survival.

In one embodiment, the invention relates to an antibody of the invention for use in a method of treating metastases or metastases of a CSC cancer.

In the context of the invention, the term "treating metastasis" encompasses the prevention of metastasis of a CSC cancer.

In another embodiment, the invention relates to an antibody of the invention to prevent relapse or recurrence of a CSC cancer.

The antibody of the invention for the treatment of CSC cancer is used to improve the survival (more particularly the 5-year survival rate) of patients affected by a CSC cancer.

Therefore, in a particular embodiment, the invention relates to an antibody of the invention for use in a method for the treatment of a CSC cancer, said CSC cancer being a cancer associated with a poor prognosis.

As used herein, a cancer associated with a poor prognosis corresponds to a cancer associated with an average prognosis of less than 5 years, preferably less than 2 years and even preferably less than 1 year. In this context, the antibody of the invention is preferably used in combination with at least one other anti-cancer compound or another anti-cancer therapy for the treatment of CSC cancer. This combination can enhance the effects of anticancer therapy and reduce the risk of spreading, relapse or recurrence of said CSC cancer.

The compounds and anticancer therapies are diverse and well known in the art. They cover conventional and general anti-cancer strategies, such as chemotherapy, radiotherapy, surgery, but also hormonal therapy. They also include more specific therapies, including targeted therapy, including monoclonal antibodies directed against tumor antigens and tyrosine kinase and CDK inhibitors, angiogenesis inhibitors ...

In one embodiment, the invention relates to an antibody of the invention and at least one other anticancer compound as a combined preparation for separate, simultaneous or sequential use in the treatment of CSC cancer. In another embodiment, the invention relates to an antibody of the invention combined with surgery, chemotherapy, hormonal therapy, target-directed therapy and / or radiotherapy for separate, simultaneous or sequential use in the treatment of CSC cancer.

The combination of an antibody of the invention with another cancer treatment could lead to complete tumor regression.

A second object of the invention relates to a pharmaceutical composition for use in a method for treating CSC cancer, said pharmaceutical composition comprising an antibody of the invention or one of its functional fragments.

The antibodies of the invention have been described above.

According to the invention, said antibody comprises:

a) a light chain comprising at least one light chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO: 3 for CDR-L3, and / or b) a heavy chain comprising at least one heavy chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, SEQ ID NO: 6 for CDR-H3.

In a particular embodiment, the antibody of the invention comprises:

a) a light chain comprising a light chain framework of an immunoglobulin light chain and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR- L2, SEQ ID NO: 3 for CDR-L3, and / or

b) a heavy chain comprising a heavy chain framework of an immunoglobulin heavy chain and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR- H2, SEQ ID NO: 6 for CDR-H3.

In another embodiment, said composition comprises a functional fragment of an antibody of the invention in which said fragment is chosen from the group comprising or consisting of Fab, Fab ', F (ab') ² , Facb, Fd, scFv, diabody and monobodies that include fragments of VHH and fragments of human VH.

According to the invention, said fragment comprises the CDRs of the antibody of the invention and is capable of recognizing the O-acetylated GD2 ganglioside.

In another embodiment, said composition comprises an antibody of the invention wherein said antibody is a chimeric antibody.

In a preferred embodiment, said antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 8.

In another embodiment, said composition comprises an antibody of the invention wherein said antibody is a humanized antibody.

In another embodiment, the antibody of the invention is an immunoconjugate.

In a particular embodiment, the antibody of the invention is an immunoconjugate in which said immunoconjugate comprises an antibody of the invention or one of its functional fragments and a cytotoxic agent.

In another particular embodiment, the antibody of the invention is an immunoconjugate in which said immunoconjugate comprises an antibody of the invention or one of its functional fragments and a radioisotope. CSC cancers of the invention have also been described above.

In one embodiment, the composition of the invention is for use in a method of treating a CSC cancer chosen from the group comprising or consisting of leukemia, including acute myeloid leukemia and acute lymphoid leukemia, breast cancer, glioma including glioblastoma, cancer Colorectal, pancreatic cancer, prostate cancer, lung cancer, liver cancer, bladder cancer or gastric cancer.

In a preferred embodiment, said CSC cancer is glioma, breast cancer, acute lymphoid leukemia or acute myeloid leukemia.

In another embodiment, said CSC cancer comprises cells that have at least one phenotype chosen from the group comprising or consisting of: CD34 + / CD38-, CD34 + / CD19-, CD34 + / CD19 +, CD34 + / CD38 + / CD19 +, CD34 + / CD38- / CD19 +, CD133 + / CD38 ", CD133 + / CD19-, interleukin-3-receiver to +, CD33 +, CD44 + / CD24 '/ low, CD44 + / CD24YESA +, CD44 + / CD24bajo / -linVALDH +, CD133 +, A2B5 +, SSEA-1 +, CD133 + / ESAalto / CD44 +, CD166 +, CD26 +, CD44 + / CD133 + / a2p1 +, CD44 +, Sca + / CD45VPecamYCD34 +, ALDH1 + / Oct4 + / CD133 + / ABCG2 + / CXCR4 +, CD133 + / CD44 +, EpCAMD / CD3 + CD6 +

In a more particular embodiment of the invention, the CSC cancer of the invention comprises CD133 + cells, CD44 + cells, more particularly CD44 + / CD24 "/ low cells or CD34 + cells, more particularly CD34 + / CD38- or CD34 + / CD19 + cells, even more particularly CD34 + / CD19 + / CD38- or CD34 + / CD19 + / CD38 + cells.

In a preferred embodiment of the invention, the CSC cancer of the invention is characterized by a subpopulation of cancer stem cells in which at least 10% of the cancer stem cells express the O-acetylated GD2 ganglioside, preferably at least 30 % of the cancerous stem cells, and more preferably at least 50% of the stem cells express the O-acetylated GD2 ganglioside.

In a particular embodiment of the invention, said CSC cancer is breast cancer. In a more particular embodiment, said CSC cancer is breast cancer in which cancer stem cells are characterized by a CD44 + / CD24 '/ low phenotype.

In another particular embodiment of the invention, said CSC cancer is glioma. In a more particular embodiment, said CSC cancer is glioma in which cancer stem cells are characterized by a CD133 + phenotype. In another particular embodiment of the invention, said CSC cancer is leukemia wherein the cancer stem cells are characterized by a CD34 + phenotype, more particularly said CSC cancer is acute myeloid leukemia where the cancer stem cells are characterized by a CD34 + / phenotype. CD38 'or acute lymphoid leukemia where cancer stem cells are characterized by a CD34 + / CD19 + phenotype, preferably a CD34 + / CD19 + / CD38- or CD34 + / CD19 + / CD38 + phenotype.

In one embodiment, the invention relates to a composition of the invention for use in a method of treating metastases or metastases of a CSC cancer.

In another embodiment, the invention relates to a composition of the invention for use in a method of preventing relapse or recurrence of a CSC cancer.

Therefore, in a particular embodiment, the invention relates to a composition of the invention for the treatment of a CSC cancer, wherein said CSC cancer is a cancer associated with a poor prognosis.

In one embodiment, the invention relates to a composition of the invention and at least one other anticancer compound as a combined preparation for separate, simultaneous or sequential use in a method for the treatment of CSC cancer.

In another embodiment, the invention relates to a composition of the invention combined with surgery, chemotherapy, hormonal therapy, target-directed therapy and / or radiotherapy for separate, simultaneous or sequential use in a method for the treatment of CSC cancer.

The pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce similar allergic or undesirable reactions, such as gastric discomfort, dizziness and the like, when administered to a human being. Preferably, as used herein, the term "pharmaceutically acceptable" means authorizable by a federal or state government regulatory agency or listed in the US Pharmacopoeia. or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.

The term "vehicle" refers to a solvent, adjuvant, excipient or carrier with which the compound is administered. Such pharmaceutical vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

The route of administration of the composition of the invention is preferably parenteral; As used herein, the term "parenteral" includes intratumoral, intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration. Therefore, the pharmaceutical composition contains vehicles that are pharmaceutically acceptable for a formulation intended to be injected. These may in particular be isotonic and sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium and the like or mixtures of said salts), or dry compositions, especially lyophilized which, by addition, depending on the case, of Sterilized water or physiological saline solution, allow the constitution of injectable solutions. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Of these, the most preferred is intravenous or intratumoral administration.

The antibody can be solubilized in a buffer or water or incorporated into emulsions, microemulsions, hydrogels (for example, hydrogels based on copolymers of PLGA-PEG-PLGA triblocks), in microspheres, in nanospheres, in microparticles, in nanoparticles (by for example, poly (lactic-co-glycolic acid), microparticles (for example, poly (lactic acid) (PLA); poly (lactic-co-glycolic acid) (PLGA)); microspheres, nanospheres, microparticles or polyglutamate nanoparticles) , in liposomes or other galenic formulations. In all cases, the formulation must be sterile and fluid to the extent of acceptable syringeability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the development of microorganisms.

The antibody can be formulated in a composition in neutral or saline form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids, such as acetic, oxalic, tartaric. , Mandelic and the like. The salts formed with the free carboxyl groups can also come from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), their suitable mixtures and vegetable oils. The conjugates of the invention can also be modified, by pegylation as an example, to increase their bioavailability.

The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be achieved by the use in the compositions of agents that delay absorption, for example, aluminum monostearate, gelatin, polyols, covalent and non-covalent formulations that improve the half-life.

There are numerous causes of instability or degradation of peptides, including hydrolysis and denaturation. Hydrophobic interaction can cause agglutination of molecules (i.e. aggregation). Stabilizers may be added to reduce or prevent such problems.

Stabilizers include cyclodextrin and its derivatives (see US Patent No. 5,730,969). Suitable preservatives, such as sucrose, mannitol, sorbitol, trehalose, dextran and glycerin can also be added to stabilize the final formulation. A stabilizer selected from ionic and non-ionic surfactants, D-glucose, D-galactose, D-xylose, D-galacturonic acid, trehalose, dextrans, hydroxyethyl starches and mixtures thereof can be added to the formulation. The addition of alkali metal salt or magnesium chloride can stabilize a peptide. The peptide can also be stabilized by contacting it with a saccharide selected from the group consisting of dextran, chondroitinsulfuric acid, starch, glycogen, dextrin and alginic acid salt. Other sugars that can be added include monosaccharides, disaccharides, sugar alcohols and mixtures thereof (eg, glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol, xylitol). The polyols can stabilize a peptide and are water miscible or water soluble. Suitable polyols may be polyhydric alcohols, monosaccharides and disaccharides including mannitol, glycol, ethylene glycol, propylene glycol, trimethyl glycol, vinyl pyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose and their polymers. Several excipients can also stabilize peptides, including serum albumin, amino acids, heparin, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinyl pyrrolidone, hydrolyzed gelatin and ammonium sulfate. In one embodiment of the invention, the pharmaceutical composition of the invention further comprises an additional active compound in separate or unit dosage form for separate, simultaneous or sequential use or administration.

A third object of the invention relates to a method for treating a CSC cancer in a subject that comprises administering to said subject in need thereof an effective amount of an antibody or a composition of the invention. As used herein, the term "subject" refers to a mammal, such as a rodent, a feline, a canine or a primate, and most preferably a human being.

The compositions and antibodies of the invention have been described in detail above.

In one embodiment, the invention relates to said method for treating a CSC cancer chosen from the group comprising or consisting of leukemia including acute myeloid leukemia and acute lymphoid leukemia, breast cancer, glioma including glioblastoma, colorectal cancer, pancreatic cancer , prostate cancer, lung cancer, liver cancer, bladder cancer or gastric cancer.

In a preferred embodiment, said CSC cancer is glioma, breast cancer, acute lymphoid leukemia or acute myeloid leukemia.

In another embodiment, said CSC cancer comprises cells that have at least one phenotype chosen from the group comprising or consisting of: CD34 + / CD38 ', CD34 + / CD19', CD34 + / CD19 +, CD34 + / CD38 + / CD19 +, CD34 + / CD38 ' / CD19 +, CD133 + / CD38-, CD133 + / CD19 ', interleukin-3-receiver a +, CD33 +, CD44 + / CD24' / low, CD44 + / CD24 '/ ESA +, CD44 + / CD24-down / -linVALDH +, CD133 +, A2B5 +, SSEA- 1+, CD133 + / ESAalto / CD44 +, CD166 +, CD26 +, CD44 + / CD133 + / a2p1 +, CD44 +, Sca + / CD45VPecamYCD34 +, ALDH1 + / Oct4 + / CD133 + / ABCG2 + / CXCR4 +, CD133 + / CD44 +, EpCAM + CD4 + P6 + CD4 + P3 + CD4 +, CD6 + .

In a more particular embodiment of the invention, the CSC cancer of the invention comprises CD133 + cells, CD44 + cells, more particularly CD44 + / CD24 '/ low cells or CD34 + cells, more particularly CD34 + / CD38- or CD34 + / CD19 + cells, even more particularly CD34 + / CD19 + / CD38- or CD34 + / CD19 + / CD38 + cells. In a preferred embodiment of the invention, the CSC cancer of the invention is characterized by a subpopulation of cancer stem cells in which at least 10% of the cancer stem cells have the O-acetylated GD2 ganglioside on their surface, preferably at less than 30% of the cancer stem cells, and more preferably at least 50% of the stem cells have the O-acetylated GD2 ganglioside on their surface. In a particular embodiment of the invention, said CSC cancer is breast cancer. In a more particular embodiment, said CSC cancer is breast cancer in which cancer stem cells are characterized by a CD44 + / CD24 '/ low phenotype.

In another particular embodiment of the invention, said CSC cancer is glioma. In a more particular embodiment, said CSC cancer is glioma in which cancer stem cells are characterized by a CD133 + phenotype. In another particular embodiment of the invention, said CSC cancer is leukemia wherein the cancer stem cells are characterized by a CD34 + phenotype, more particularly said CSC cancer is acute myeloid leukemia where the cancer stem cells are characterized by a CD34 + / phenotype. CD38- or acute lymphoid leukemia in which cancer stem cells are characterized by a CD34 + / CD19 + phenotype, preferably a CD34 + / CD19 + / CD38- or CD34 + / CD19 + / CD38 + phenotype.

In one embodiment, the invention relates to said method for treating metastases or metastases of a CSC cancer. In another embodiment, the invention relates to said method for preventing relapse or recurrence of a CSC cancer. In a particular embodiment, the invention relates to a method for treating a CSC cancer of the invention, wherein said CSC cancer is a cancer associated with a poor prognosis.

In one embodiment, the invention relates to a method for treating a CSC cancer in a subject comprising administering to said subject in need an effective amount of an antibody or a composition of the invention and at least one other anti-cancer compound as a preparation. combined for separate, simultaneous or sequential use in the treatment of CSC cancer.

In another embodiment, the invention relates to a method for treating CSC cancer of the invention combined with surgery, chemotherapy, hormonal therapy, target-directed therapy and / or radiation therapy for separate, simultaneous or sequential use in the treatment of cancer. of CSC.

An "effective amount" of the composition is an amount that is sufficient to induce tumor growth regression. The doses used for administration can be adapted according to various parameters, in particular depending on the mode of administration used, the relevant pathology or, alternatively, the desired duration of treatment. Naturally, the form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend on the condition to be treated, the severity of the disease, the age, the weight and the sex of the subject, etc. The effective dose ranges provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dose can be adapted to the individual subject, as understood and can be determined by one skilled in the art, without excessive experimentation.

As an illustration, an effective amount of at least one conjugate is from about 50 to about 1,000 mg / m2, more preferably from about 100 to about 750 mg / m2, and most preferably from about 250 to about 500 mg / m2. Other dosages are viable, since the molecular weight of the conjugate thereof can influence it. The person skilled in the art will easily decide on a suitable dosage that is within the intervals, or if necessary, outside the intervals.

Diagnostic strategies of the invention

A fourth object of the invention relates to a method for diagnosing a CSC cancer in a subject, wherein said method comprises determining the expression of the O-acetylated GD2 ganglioside and wherein an expression of the O-acetylated GD2 ganglioside is indicative of a CSC cancer

The invention encompasses in vivo and in vitro methods .

In one embodiment, the invention relates to an in vitro method for diagnosing a CSC cancer in a subject, wherein said method comprises the step of analyzing a biological sample obtained from said subject (i) by determining the expression of the GD2 O- ganglioside. acetylated, and where an expression of the O-acetylated GD2 ganglioside in said biological sample is indicative of a CSC cancer.

Advantageously, the determination step (i) is evaluated by an antibody or its functional fragment as previously described. Said antibody or its fragment may be labeled (for example, a radiolabeled antibody, labeled with a chromophore, labeled with a fluorophore or labeled with enzyme) or be a derivative (by for example, an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein / ligand couple (for example, biotin-streptavidin)).

Such analysis can be evaluated by a variety of methods well known to those skilled in the art, including, but not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunosorbent assay ( ELISA), thin layer immunochromatography (ITLC), imaging techniques, in particular positron emission tomography (PET-Scan). The in vivo methods of the invention are evaluated by an antibody of the invention or its functional fragment, which is suitable for in vivo administration . Preferably, said in vivo methods are evaluated by in vivo imaging methods , such as the PET-Scan method. Such methods are well known in the art. As used herein, the term "biological sample" means any biological sample from a patient, preferably said biological sample refers to a biopsy that has been removed from the body of a subject before executing the method of the invention.

In one embodiment, said CSC cancer is chosen from the group comprising or consisting of leukemia including acute myeloid leukemia and acute lymphoid leukemia, breast cancer, glioma including glioblastoma, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer. , liver cancer, bladder cancer or gastric cancer.

In a preferred embodiment, said CSC cancer is a glioma, a breast cancer, an acute lymphoid leukemia or an acute myeloid leukemia.

A fifth object of the invention relates to the use of the G-2 O-acetylated ganglioside as a biomarker of CSC cancer.

The term "O-acetylated GD2 ganglioside" refers to a ganglioside derived from the GD2 ganglioside and corresponding to 9 (7) -O-acetyl-GD2.

A sixth object of the invention relates to a method for predicting the response of a subject affected by CSC cancer to a treatment with an antibody or a composition of the invention, wherein said method comprises detecting the presence of cells expressing the ganglioside. O-acetylated GD2 in a biological sample of said subject.

In one embodiment, the invention relates to said method for predicting the response of a subject affected by CSC cancer to a treatment with an antibody or a composition of the invention, wherein the presence of cells expressing the O-acetylated GD2 ganglioside in a biological sample of said subject it is correlated with a good probability that said subject responds to said treatment.

In a more particular embodiment, the invention relates to said method which comprises detecting the level of cancer stem cells expressing the G-2 O-acetylated ganglioside among all cancer stem cells in a biological sample of said subject.

In a preferred embodiment, a level of at least 10%, particularly at least 30%, preferably at least 50% of the cancerous stem cells presenting the G-2 O-acetylated ganglioside on its surface among all the cancerous stem cells in a biological sample of said subject correlates with a high probability that said subject responds to said treatment.

In one embodiment, said biological sample is a cancer sample, preferably a CSC cancer sample.

In one embodiment, said CSC cancer is chosen from the group comprising or consisting of leukemia including acute myeloid leukemia and acute lymphoid leukemia, breast cancer, glioma including glioblastoma, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer. , liver cancer, bladder cancer or gastric cancer.

In a preferred embodiment, said CSC cancer is a glioma, a breast cancer, an acute lymphoid leukemia or an acute myeloid leukemia.

In one embodiment of the invention, said method comprises detecting the presence of cells expressing the O-acetylated GD2 ganglioside in patients by non-invasive methods such as imaging using an in vivo imaging agent . Such methods are well known in the art.

Examples

In the following, the invention is described in more detail with reference to amino acid sequences, nucleic acid sequences and examples. However, no limitation of the invention is intended by the details of the examples. Rather, the invention relates to any embodiment that comprises details that are not explicitly mentioned in the examples of said invention, but which experts find without undue effort.

• Gliomas

Evaluation of ganglioside OAcGD2 expression in glioma

° Expression of OAcGD2 in glioblastoma biopsies

The inventors evaluated the expression of the O-acetylated GD2 ganglioside (OAcGD2) in 22 glioblastoma samples using immunohistochemistry (IHC). An example of immunohistochemical staining for diagnosis is shown in Figure 1, where the tissues are stained for the OAcGD2 antigen.

Tumor samples (glioblastomas) were obtained by surgical excision. A tissue sample (volume <0.5 cm3) was removed and frozen in cooled isopentane to the temperature of liquid nitrogen. After 60 seconds, the sample was removed and transferred and kept at -70 ° C. 10 pm cuts were made using a cryostat. The cuts were collected on SUPERFROST GOLD (VWR) glass slides and air dried for 3 minutes. Then they were fixed in acetone (-20 ° C) for 10 minutes and air dried again. The cuts were then stored at -20 ° C until use.

The immunoreactivity of the 8B6 antibody was detected by incubation in stages with a biotinylated goat anti-mouse antibody, followed by streptavidin-biotin-peroxidase complex and DAB substrate. The sample was analyzed by a pathologist with an optical microscopy equipment and classified as negative, 1+, 2+ or 3+, compared to a frozen human neuroblastoma cut as a positive control and tissue incubated with an irrelevant IgG3 antibody as a negative control. . The results are summarized in Table 2.

Table 2

(ND: Not determined)

They found that all samples were positively stained with the anti-OAcGD2 (mAb) 8B6 monoclonal antibody, with an IHC score ranging from 1+ to 3+. In 16 samples among 22, 100% of the tumor cells were positive within the tumor.

° Expression of OAcGD2 in glioma cell lines and primary cells

The expression profiles of CD133 and OAcGD2 in U87MG human glioblastoma cells were determined by flow cytometry analysis. The cells were stained with the mouse precursor mAb 8B6 specific for OAcGD2 and with the CD133-FITC antibody. The cells were washed 3 times with ice-cold PBS, incubated with 8B6 mAb (10 pg / mL in 1% PBS-BSA) for 30 minutes at 4 ° C. After 3 washes with ice-cold PBS, the bound primary antibody was detected with the F (ab) ' ² fragment of the goat anti-mouse secondary antibody conjugated to FITC for 30 minutes. After washing, the cells were incubated with the CD133-APC conjugate antibody for 30 minutes at 4 ° C. After washing the cells 3 times with ice-cold PBS, the cells were analyzed with a FACSCalibur (BD) cytometer using the Cell Quest Pro (BD) software. An antibody of the same isotype was used as a negative control. At least 1 x 104 episodes in cell lines were measured. Using flow cytometry, the inventors also detected the OAcGD2 antigen on the cell surface of human glioma cell lines (3/3) and human primary glioma cells (12/12). For human glioma cell lines, the rate of OAcGD2 positive cells varied from 61 to 85% (Figure 2, Table 3).

They also observed the presence of CD133 + OAcGD2 + CSC in these glioma cell lines, and within CD133 positive cells, the rate of OAcGD2 positive cells was approximately 85 to 98%.

For human primary glioma cells, the rate of OAcGD2 positive cells varied between 32 and 94%. They also observed the presence of CD133 + OAcGD2 + CSC in these primary glioma cells, and within CD133 positive cells, the rate of OAcGD2 positive cells was approximately 62 to 100% (Table 3).

Table 3. OAcGD2 +, CD133 + and OAcGD2 + CD133 + populations (percentage of OAcGD2 + cells among CD133 +) in glioma cells

The results confirm those obtained in glioblastoma biopsies with a strong expression of OAcGD2 in primary glioma cells and also in glioma cell lines. In addition, the search for CSC (CD133 +) between these primary glioma cells and also the glioma cell lines has allowed the identification of several percentages of said CSC (CD133 +). Now, and surprisingly, the results have shown that the expression of OAcGD2 is enriched in these CSCs compared to non-CSCs. As an example, the percentage of OAcGD2 positive cells was double for CSC than for non-CSC (data not shown).

Finally, the results established that OAcGD2 is enriched in CSC of gliomas and can be used to facilitate the use of these cells as a target.

Effects of mAb 8B6 on glioma cells expressing OAcGD2.

U87-MG tumor cells (5 x 105 cells) were seeded in 12-well flat bottom plates and incubated with 8B6 mAb or control IgG3 antibody for 24 hours at 37 ° C, 5% CO ² . Images of the cell cultures were obtained with a LEICA DFC295 digital camera coupled to a LEICA 164 microscope.

After an incubation period of 24 hours, apoptotic cells evaluated by morphological changes were observed under a phase contrast microscope with the same magnification, 200. The arrow indicates the apoptotic cell.

Treatment with mAb 8B6 induced changes in the morphology of U87MG cells. Cells incubated with mAb 8B6 had a spherical shape and formed blisters, which loosely bound to the bottom of the culture vessel or floated in the culture media. Segregated cells in several fragments are characteristic of apoptosis (Figure 3). Among U87-MG cells, it can be speculated that CSCs, whose cells expressed the highest percentage of OAcGD2, die after apoptosis.

Effects of mAb 8B6 on glioblastoma in an in vivo model

Female nude (nu / nu) mice (Harlan Laboratories) were cared for and maintained in accordance with European Animal Welfare standards under an approved institutional protocol for animal care and use in an animal facility at the University of Nantes accredited by The French Ministry of Agriculture.

Aliquots of suspension of U251 cells (3 x 106 cells / 100 pL containing an equal volume of RPMI and matrigel) were implanted subcutaneously on the left side of the mice. One week later, the tumor mass was present in all originally injected mice that were then randomized into three equal groups and antibody treatment was initiated. Anti-OAcGD2 mAb 8B6 was formulated as a phosphate buffered saline solution and injected intravenously. One group was treated with a 500 pg injection of 8B6 mAb while the control group was treated with an equal volume of PBS only as a vehicle. The third group was treated with an isotype mAb thereof as a negative control. Tumors were measured with calipers; The tumor volume was calculated according to the formula: length x width2 x n / 6.

The inventors also found that anti-OAcGD2 mAb inhibits the development of human glioblastoma tumors in mice (Figure 4). When the tumor volume reached 100 mm3, the mice were injected with either 500 pg of 8B6 mAb or control IgG3 mAb and tumor development was followed. The 8B6 antibody inhibited the development of human U251 glioblastoma tumor compared to mice treated with control IgG3 and with vehicle. Sixty-eight days after antibody injection, tumor volumes had an average volume of 423 ± 236 mm3 and 405 ± 176 mm3 in the vehicle-treated and control IgG3 groups, respectively. In contrast, treatment with mAb 8B6 inhibited the development of the U251 tumor with an average tumor volume of 254 ± 142 mm3. The specificity of the treatment was demonstrated since treatment with an equivalent amount of non-specific IgG3 antibody remained ineffective. Finally, it was shown that non-CSC cells can be identified in mice treated with 8B6 compared to mice treated with IgG3 (data not shown).

In conclusion, the results established that the anti-OAcGD2 antibody has allowed the removal of CSC cells in vivo, this elimination being associated with a clear and strong inhibition of tumor development.

• Breast cancers

Evaluation of ganglioside OAcGD2 expression in breast cancer

° Expression of OAcGD2 in breast cancer tissues

The inventors have performed a preliminary immunohistochemistry to evaluate the expression of OAc-GD2 in cancer tissues embedded in paraffin and formed with formalin with the monoclonal antibody 8B6.

Then, they established that, among 25 stained breast cancer biopsies, 18 samples were positively stained, presenting 1 sample with a weak staining (score 1+), presenting 9 samples with moderate staining (score 2+) and presenting 8 samples with strong staining (score 3+).

This strong correlation between breast cancer tissue and OAcGD2 was confirmed by other immunochemical tests performed on 28 frozen cuts of other breast cancer tissues. For this series of experimentation, all samples were positively stained for OAcGD2.

Consequently, it seems that OAcGD2 can be used as a marker for breast cancer.

° Expression of OAcGD2 in breast cancer cell lines

The expression of OAcGD2 has been evaluated in the SUM159 breast cancer cell line. The expression profile of OAcGD2 in human breast cancer stem cells was determined by flow cytometry analysis after triple fluorescent staining. Cells were incubated with primary antibodies 8B6, 14G2a and 7H2 (at 10 | jg / mL) for 45 minutes on ice in 1% PBS-BSA. After 3 washes with ice-cold PBS, primary antibody bound with goat anti-mouse IgG Alexa Fluor® 568 (H + L) (Life Technologies) was detected for 45 minutes on ice. The cells were then fixed to 4% PFA for 10 minutes and then incubated for 25 minutes with anti-CD24 conjugate with FITC and anti-CD44 conjugate with APC (BD). After washing, the cells were analyzed with an LSRII flow cytometer (BD) using the FlowJo (BD) software. At least 1 x 104 episodes in the cell lines were measured.

It was found that approximately 35% of the cells expressed OAcGD2 (antibody 8B6), while only about 15% expressed the ganglioside GD2 (antibody 14G2a). Said different expression was confirmed in other breast cancer cell lines (HMLE, data not shown). Then, the proportion of CD44 + CD24 '/ low cells within the SUM159 cell line was evaluated and 94% of the cells were shown to have a CSC phenotype. If the percentage of OAcGD2 positive cells among CSCs compared to non-CSCs was not determined in SUM159, it was shown that said percentage was 5 times higher in other breast cancer cell lines (HMLE, data not shown). Therefore, and as for the results obtained in gliomas, it appears that OAcGD2 is enriched in the CSC of breast cancers and can be used to facilitate the use of these cells as a target. In addition, these results are very interesting since they allow considering the therapeutic efficacy of a treatment directed to OAcGD2 in contrast to a treatment directed to GD2. In fact, using the small percentage of cells expressing GD2 as a target does not guarantee limiting the spread of associated cancer.

Inhibition of the viability of breast cancer cells with the 8B6 antibody

The inventors performed a cell proliferation assay with anti-OAcGD2 and anti-GD2 antibodies of the SUM159 breast cancer cell line.

104 SUM159 cells (100 jL) were incubated in a 96-well microplate 24 hours at 37 ° C. Antibodies of 80-1.25 jg / mL in 50 jL of medium were added and incubated 24 hours at 37 ° C. 50 jg of MTT was added to each well and incubated at least 4 hours at 37 ° C, before cells were solubilized with 10% ^s D ^s to stop cell conversion of the MTT dye and incubated ON at 37 ° C. Then the absorbance at 570 and 650 nm was read. The absorbance of the product at 650 nm was subtracted from the absorbance at 570 nm (Abs ⁵⁷⁰ -Abs ⁶⁵⁰ ) to calculate the total dye conversion. Four control wells with cells treated with 20 jg of etoposide provide the blank for absorbance giving 0% viability. Viability (%) was expressed as a percentage in relation to untreated cells and each value is represented in quadruplicate as mean ± SEM.

The 8B6 antibody induced a direct cytotoxicity in this cell line that approximates 30% inhibition of viability (Figure 5).

• Lung cancers

Evaluation of OAcGD2 ganglioside expression in small cell lung cancer

° Expression of OAcGD2 in lung cancer cell lines

The expression of OAcGD2 was evaluated in the H196 lung cancer cell line. The expression profile of OAcGD2 in human lung cancer stem cells was determined by flow cytometry analysis after double fluorescent staining. Cells were incubated with primary antibodies 8B6, 10B8 and 7H2 (at 10 | jg / mL) for 45 minutes on ice in 1% PBS-BSA. After 3 washes with ice-cold PBS, the bound primary antibody was detected with goat anti-mouse IgG conjugated to FITC (H + L) (JacksonResearch) for 45 minutes on ice. The cells were then fixed to 4% PFA for 10 minutes and then incubated for 25 minutes with an anti-CD133 conjugate with APC (Miltenyi Biotec). After washing, the cells were analyzed with a FACScalibur (BD) flow cytometer using the CellQuest (BD) software. At least 1 x 104 episodes in the cell lines were measured.

The results are presented in Figure 6.

Again, the results show that OAcGD2 and CD133 expressions were correlated with more than 50% of the CSC cells expressing OAcGD2. GD2 expression in these cells was slightly greater than the negative control (i.e. 7%).

Again, it appears that OAcGD2 is enriched in CSCs of small cell lung cancers and can be used to facilitate the use of these cells as a target.

• Leukemia

The inventors have performed a preliminary indirect immunofluorescence assay to evaluate the expression of OAc-GD2 in acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) with the 8B6 monoclonal antibody.

Two of the ALL cell lines analyzed were positive. A sample of AML from a patient was also positive.

Claims (10)

1. A pharmaceutical composition for use in the treatment of breast cancer comprising cancer stem cells that have at least one phenotype chosen from the group comprising or consisting of CD44 + / CD24 '/ low, CD44 + / CD24' / low / ESA + or CD44 + / CD24 '/ bass / linYALDH1 +, said pharmaceutical composition comprising an antibody that recognizes the O-acetylated GD2 ganglioside or one of its functional fragments, and wherein said antibody comprises: a) a light chain comprising at least one light chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO: 3 for CDR-L3, and / or b) a heavy chain comprising at least one heavy chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, SEQ ID NO: 6 for CDR-H3. 2. The pharmaceutical composition for use in the treatment of breast cancer according to claim 1, wherein said antibody is a functional fragment of an antibody of claim 1, wherein said fragment is chosen from the group comprising or consisting of Fab , Fab ', F (ab') ² , Facb, scFv, diabodies that include fragments of VHH and fragments of human VH. 3. The pharmaceutical composition for use in the treatment of breast cancer according to claim 1, wherein said antibody is a chimeric or humanized antibody. 4. The pharmaceutical composition for use in the treatment of breast cancer according to claim 1 or 3, wherein said antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 8. 5. The pharmaceutical composition for use in the treatment of breast cancer according to any one of claims 1 to 4, wherein said breast cancer is characterized by a subpopulation of cancer stem cells in which at least 10% of the cells Cancerous mothers express the GD2 O-acetylated ganglioside. 6. A combined preparation comprising the antibody that recognizes the O-acetylated GD2 ganglioside, or one of its functional fragments, as defined in any one of claims 1 to 4, and at least one other anticancer compound for separate, simultaneous or sequential in the treatment of breast cancer. 7. The pharmaceutical composition for use in the treatment of breast cancer according to any one of claims 1 to 5, which is combined with surgery, chemotherapy, hormonal therapy, target-directed therapy and / or radiotherapy for separate, simultaneous or simultaneous use. sequential in the treatment of breast cancer. 8. An in vitro method for diagnosing breast cancer comprising cancer stem cells that have at least one phenotype chosen from the group comprising or consisting of CD44 + / CD24- / low, CD44 + / CD24- / low / ESA + or CD44 + / CD24 '/ low / linVALDH1 in a subject, wherein said method comprises the step of analyzing a biological sample obtained from said subject (i) by determining the expression of the O-acetylated GD2 ganglioside using an antibody that recognizes the O-acetylated GD2 ganglioside, or one of its functional fragments, wherein said antibody comprises: a) a light chain comprising at least one light chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO: 3 for CDR-L3, and / or b) a heavy chain comprising at least one heavy chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, SEQ ID NO: 6 for CDR-H3, and wherein an expression of the O-acetylated GD2 ganglioside in said biological sample is indicative of breast cancer. 9. Use of an antibody that recognizes the O-acetylated GD2 ganglioside or one of its functional fragments, wherein said antibody comprises: c) a light chain comprising at least one light chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO: 3 for CDR-L3, and / or d) a heavy chain comprising at least one heavy chain variable region framework of an immunoglobulin and three complementarity determining regions (CDRs) defined by the sequences SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, SEQ ID NO: 6 for CDR-H3, to detect the G-2 O-acetylated ganglioside as a breast cancer biomarker comprising cancer stem cells that have at least one phenotype chosen from the group comprising cD44 + / CD24 '/ low, CD44 + / CD24' / low / ESA + or CD44 + / CD24- / bajt7linVALDH1 +. 10. A method for predicting the response of a subject affected with breast cancer to a treatment with an antibody that recognizes the O-acetylated GD2 ganglioside, or one of its functional fragments, as defined in any one of claims 1 to 4 , wherein said method comprises detecting the presence of cancer stem cells expressing the G-2 O-acetylated ganglioside in a biological sample of said subject.